Cyclophilin-D (Cyp-D) is a peptidyl prolyl isomerase which catalyzes the cis-trans isomerization of peptidyl prolyl bonds and thereby regulates conformational changes of target proteins. Cyp-D is the only cyclophilin res- ident in mitochondria, and until recently it was best known for its effect on the mitochondrial permeability transi- tion pore. We recently discovered that Cyp-D interacts with two of the three mitochondrial transcription factors (i.e., TFB1M and TFB2M but not TFAM) and subsequently found that Cyp-D also interacts with the mitochon- drial RNA polymerase. Genetic silencing of Cyp-D in isolated cells disrupted mitochondrial gene expression re- sulting in a reduction in mitochondrial O2 consumption (VO2). More recently, working with a constitutive Cyp-D knock out (Cyp-D KO) mice, we showed also in vivo reductions in VO2 (at rest and exercise). Concomitantly, the respiratory exchange ratio increased suggesting a metabolic shift favoring utilization of carbohydrates over fat. Intriguingly, the exercise capacity was increased pointing to an adaptive response whereby O2 utilization efficiency was increased. Using in silico modeling, we have identified small molecules able to destabilize the interaction between Cyp-D and TFB2M. Thus, we hypothesize that Cyp-D plays a key role in oxygen me- tabolism by regulating mitochondrial gene expression and signaling an adaptive increase in oxygen utilization efficiency. We propose to further this hypothesis based on two specific aims. Specific Aim 1: De- signed to complete the characterization of the Cyp-D effects on mitochondrial gene expression using cardiac relevant tissue and examine additional downstream processes ? also mediated by Cyp-D ? that may impact cell respiration (structured in 4 Sub Aims). Sub-Aim 1, designed to completely characterize the interactions among key players of the mitochondrial transcription machinery and assess their dependency on Cyp-D PPI- ase activity; Sub-Aim 2, designed to confirm that the effects of Cyp-D on mitochondrial gene expression are HSP2 specific (as suggested by preliminary data) and to assess whether Cyp-D silencing could signal up- stream and affect the expression of nuclear-encoded proteins of respiratory complexes; Sub-Aim 3, designed to confirm that Cyp-D silencing reduces mitochondrial encoded subunits of the respiratory chain complexes and F1FoATP synthase and examine whether Cyp-D overexpression results in the opposite effect; and Sub- Aim 4, to investigate whether Cyp-D also acts as chaperone aiding in the assembly of respiratory complexes. Studies under Specific Aim 1 will be conducted in adult mouse primary cardiomyocytes, fibroblasts, and tissues from Cyp-D KO and wild-type (WT) mice when relevant. Cyp-D will be modulated by using lentiviral vectors harboring constructs to silence or overexpress Cyp-D and pharmacological inhibition using cyclosporine A (CsA) and newly identified small molecules. Specific Aim 2: Designed to determine the functional conse- quences of genetic ablation or pharmacological inhibition of Cyp-D in mice and examine the underlying adap- tive mechanisms leading to increased O2 utilization efficiency. Cyp-D activity will be reduced by using constitu- tive and conditional Cyp-D KO mice and by pharmacological inhibition with cyclosporine A and small molecules able to selectively destabilize the interaction between Cyp-D and TFB2M. With constitutive Cyp-D KO mice, we will assess whether the findings in cell systems after acute Cyp-D ablation also occur in vivo and examine the adaptive responses leading to increased O2 utilization efficiency. With conditional Cyp-D KO mice, we will as- sess the immediate effects of Cyp-D ablation and whether an adaptive response occurs, subsequently examin- ing whether similar effects can be elicited through pharmacological inhibition. Myocardial energy effects along with functional myocardial effects will be assessed using 31P magnetic resonance spectroscopy and magnetic resonance imaging. Understanding the underlying mechanisms of increase O2 utilization efficiency may have important implications for human physiology and disease and plan to also examine effects in acute conditions of reduced O2 availability using models of cardiac arrest and hemorrhagic shock.